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, 385 (Pt 1), 11-20

Engineering ML-IAP to Produce an Extraordinarily Potent Caspase 9 Inhibitor: Implications for Smac-dependent Anti-Apoptotic Activity of ML-IAP

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Engineering ML-IAP to Produce an Extraordinarily Potent Caspase 9 Inhibitor: Implications for Smac-dependent Anti-Apoptotic Activity of ML-IAP

Domagoj Vucic et al. Biochem J.

Abstract

ML-IAP (melanoma inhibitor of apoptosis) is a potent anti-apoptotic protein that is strongly up-regulated in melanoma and confers protection against a variety of pro-apoptotic stimuli. The mechanism by which ML-IAP regulates apoptosis is unclear, although weak inhibition of caspases 3 and 9 has been reported. Here, the binding to and inhibition of caspase 9 by the single BIR (baculovirus IAP repeat) domain of ML-IAP has been investigated and found to be significantly less potent than the ubiquitously expressed XIAP (X-linked IAP). Engineering of the ML-IAP-BIR domain, based on comparisons with the third BIR domain of XIAP, resulted in a chimeric BIR domain that binds to and inhibits caspase 9 significantly better than either ML-IAP-BIR or XIAP-BIR3. Mutational analysis of the ML-IAP-BIR domain demonstrated that similar enhancements in caspase 9 affinity can be achieved with only three amino acid substitutions. However, none of these modifications affected binding of the ML-IAP-BIR domain to the IAP antagonist Smac (second mitochondrial activator of caspases). ML-IAP-BIR was found to bind mature Smac with low nanomolar affinity, similar to that of XIAP-BIR2-BIR3. Correspondingly, increased expression of ML-IAP results in formation of a ML-IAP-Smac complex and disruption of the endogenous interaction between XIAP and mature Smac. These results suggest that ML-IAP might regulate apoptosis by sequestering Smac and preventing it from antagonizing XIAP-mediated inhibition of caspases, rather than by direct inhibition of caspases.

Figures

Figure 1
Figure 1. Protein amino acid sequences and constructs
(A) Amino acid sequence alignment of MLBIR and XIAP-BIR3. The secondary structure of MLBIR is indicated above the sequence. Residues indicated with an asterisk are within 4 Å of the Smac-based peptide in the MLBIR–AVPIAQKSE complex structure [15]. Sequence identity between the wild-type ML-IAP construct and XIAP-BIR3 is indicated. MLBIR-Q corresponds to a seven residue C-terminal truncation of MLBIR (i.e. the C-terminal residue of MLBIR-Q is Gln172). The residues shown in bold italics in the sequence of XIAP-BIR3 are those that were exchanged into MLBIR-Q to produce the chimeric constructs MLXBIR3 and MLXBIR3SG. (B) Schematic representation of the chimeric variants. Residues present in wild-type MLBIR-Q are grey, whereas those present in wild-type XIAP-BIR3 are black. (C) Schematic representation of double and triple point mutants.
Figure 2
Figure 2. Caspase 9 binding by BIR domain constructs
293T cells were transiently transfected with caspase 9 (Casp-9) and FLAG-tagged BIR domain proteins or vector. After 40 h, cells were lysed in Nonidet P40 lysis buffer (120 mM Tris/HCl, 150 mM NaCl, 1% Nonidet P40, 1 mM DTT and protease inhibitor cocktail) and lysates immunoprecipitated (IP) with anti-FLAG antibody. Samples were then immunoblotted (W) with anti-caspase 9 and anti-FLAG antibodies. (A) Comparison of chimeric proteins MLXBIR3 and MLXBIR3SG with wild-type MLBIR and XIAP-BIR3. (B) Comparison of double and triple mutant MLBIR-Q constructs with wild-type MLBIR. (C) Comparison of XIAP-BIR3 triple mutant with wild-type XIAP-BIR3.
Figure 3
Figure 3. Anti-apoptotic effect of BIR domain constructs
MCF7 cells were transiently transfected with the reporter plasmid pCMV-βgal and either vector control alone or BIR domain constructs. Following transfection, cells were exposed to doxorubicin (adriamycin; 0.5 μg/ml), stained with X-gal and apoptosis assessed by counting live and dead transfected cells. The percentage apoptosis represents the mean±S.D. for at least four sample points and is representative of three independent experiments. Asterisks designate that the difference between these two values is statistically significant (P<0.05). The relative transfection levels were evaluated by immunoblotting with anti-FLAG antibody.
Figure 4
Figure 4. Binding of ML-IAP, XIAP and their chimeric proteins to Smac
(A) MLXBIR3 and MLXBIR3SG chimeric proteins interact with mature Smac. HEK-293T cells were transiently transfected with FLAG-tagged Smac and Myc-tagged BIR domain proteins or vector control. After 40 h, cells were lysed in Nonidet P40 lysis buffer (120 mM Tris/HCl, 150 mM NaCl, 1% Nonidet P40, 1 mM DTT and protease inhibitor cocktail) and lysates were immunoprecipitated (IP) with anti-Myc antibody. Samples were then immunoblotted (W) with anti-FLAG and anti-Myc antibodies. FL-SMAC designates full-length Smac protein, and SMAC designates the mature processed protein that is produced as a result of overexpression [18]. (B) Over-expression of ML-IAP diminishes the XIAP–Smac interaction. HEK-293T cells were transiently transfected with vector control (−) or increasing amounts of Myc–ML-IAP (1 μg and 4 μg) and treated 20 h later with 1.5 μg/ml of doxorubicin. Cells were lysed 40 h following transfection in Nonidet P40 lysis buffer and lysates were immunoprecipitated with control anti-FLAG (ct) or anti-XIAP (X) antibodies. Samples were then centrifuged, and the supernatant re-immunoprecipitated with anti-Myc antibody (2nd IP). Following extensive washing with lysis buffer, immunoprecipitates and portions of total cell lysates were subjected to SDS/PAGE and immunoblotted with anti-Smac, anti-XIAP and anti-Myc antibodies.
Figure 5
Figure 5. Crystal structure of the MLXBIR3SG–peptide complex
(A) Schematic representation of the crystal structure of MLXBIR3SG in complex with the AVPIAQKSE peptide. The protein is shown in ribbon representation and the first four residues of the bound peptide are depicted in stick form, coloured by atom type; the bound zinc atom is shown as a sphere. Major secondary structure elements of the protein are labelled. The chimeric portions of the protein (Gly150 and helix-5) are coloured red. (B) The structure of wild-type MLBIR bound to the AVPIAQKSE peptide (PDB code 1OXQ; [15]) is shown in the same orientation as (A). (C) The structure of XIAP-BIR3 bound to Smac (PDB code 1G73; [14]) is shown in the same orientation as (A). Only the first four residues of Smac are depicted, in stick form. (D) Stereoview of the superposition of the peptide-binding sites of wild-type MLBIR and the chimeric protein MLXBIR3SG. The backbone of the MLXBIR3SG chimera is shown in a ribbon representation. Side chains of MLXBIR3SG and wild-type MLBIR that are within 3.8 Å of the peptide are shown in stick representation, as is the AVPI portion of the AVPIAQKSE peptide from both complexes. The MLXBIR3SG–AVPIAQKSE complex is coloured as in (A), whereas the wild-type MLBIR complex is coloured blue and depicted with thinner sticks.
Figure 6
Figure 6. XIAP-BIR3–caspase 9 interface
The structure of the XIAP-BIR3–caspase 9 complex (PDB code 1NW9; [26]) is shown; the backbone of XIAP-BIR3 is shown in ribbon representation (coloured gold) and selected side chains are shown in stick form (coloured by atom type, with gold carbons). Caspase 9 is shown as a molecular surface, except for the N-terminal peptide binding XIAP-BIR3; the first four residues of this peptide are shown in stick form (coloured by atom type, with pink carbons). Selected side chains from a superposed structure of wild-type MLBIR are shown in stick form (coloured by atom type, with blue carbons). Amino acid labels correspond to XIAP/ML-IAP residues. The side chain conformations of the ML-IAP residues Gln167 and Glu168 have been adjusted to mimic the conformations of the equivalent XIAP residues.

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